Gene therapy in transplantation: pathological consequences of unavoidable plasmid contamination with lipopolysaccharide
Gene therapy in transplantation: pathological consequences of unavoidable plasmid contamination with lipopolysaccharide
复制标题
DOI：
10.1016/s0966-3274(99)80024-0
复制
发表时间：
1999-06-01
影响因子：
1.5
通讯作者：
Orosz, CG
中科院分区：
文献类型：
--
作者：
Gordillo, GM;Xia, DY;Orosz, CG
实验研究评估了小鼠心脏移植受者对高水平和低水平脂多糖 (LPS) 污染质粒 DNA 制剂的反应。在移植前，通过向股四头肌注射编码鼠白细胞介素（IL）-4基因和β-半乳糖苷酶（β-gal）基因的质粒来转染移植受者。用仅编码β-gal基因的质粒转染的移植受体作为阴性质粒对照。用含有高水平污染 LPS 的质粒转染三组小鼠：(a) 未移植的 C57B1/6 小鼠，(b) C57B1/6 心脏同种移植受者，(c) DBA/2 (H-2(d)) --> C57BL/6 (H-2(b)) 心脏同种异体移植受者。出乎意料的是，在转染 IL-4 的同种移植物和同种异体移植物受体中观察到 24 小时内移植失败，但在单独转染 β-gal 基因的小鼠中却没有观察到移植失败。然而，组织病理学结果，例如，两个治疗组的心脏移植物中血管细胞粘附分子-1 (VCAM-1) 表达和单核肺浸润非常相似，并且与 LPS 诱导的病理学一致。 LPS 测定用于评估四种不同的质粒纯化方法的 LPS 污染程度。确定了降低 LPS 污染水平的成功策略，并在接受 LPS 接种的心脏同种异体移植受者中重复进行转染实验，所有治疗组的接种量均已最小化并标准化（6.4 EU/小鼠）。尽管接受的 LPS 水平明显较低，但在所有治疗组中，均存在持续的心脏移植物内皮细胞活化，表现为 VCAM-1 表达和持续（尽管不那么严重）的肺部病理学。我们发现，LPS 的质粒污染是不可避免的，即使非常低的水平也会改变移植受者的免疫反应，从而混淆数据解释。因此，在利用质粒 DNA 进行基因转移的实验中，特别是在免疫和炎症实验模型中，必须考虑 LPS 污染。
Experimental studies evaluated the responses of murine cardiac graft recipients to high and low levels of lipopolysaccharide (LPS) contaminating plasmid DNA preparations. Immediately prior to transplantation, graft recipients were transfected by injecting the quadriceps muscles with plasmids that encoded the murine interleukin (IL)-4 gene and P beta-galactosidase (beta-gal) gene. Graft recipients transfected with plasmids encoding only the beta-gal gene served as negative plasmid controls. Three groups of mice were transfected with plasmids containing high levels of contaminating LPS: (a) nontransplanted C57B1/6 mice, (b) C57B1/6 cardiac isograft recipients, (c) DBA/2 (H-2(d)) --> C57BL/6 (H-2(b)) cardiac allograft recipients. Unexpectedly, graft failure within 24 h was observed in IL-4 transfected isograft and allograft recipients, but not in mice transfected with the beta-gal gene alone. However, histopathological findings, for example, vascular cell adhesion moelcule-1 (VCAM-1) expression in cardiac grafts and mononuclear lung infiltration, were remarkably similar for both treatment groups and consistent with LPS-induced pathology. LPS assays were used to evaluate four different methods of plasmid purification for degree of LPS contamination. A successful strategy for reducing levels of LPS contamination was identified and transfection experiments repeated in cardiac allograft recipients receiving LPS inoculum that were minimized and standardized (6.4 EU/mouse) for all treatment groups. Despite receiving substantially lower levels of LPS, in all treatment groups there was persistent cardiac graft endothelial cell activation manifested by VCAM-1 expression and persistent, albeit less severe, lung pathology. We found that plasmid contamination with LPS was unavoidable and that even very low levels can alter immune responses in transplant recipients confounding data interpretation. Thus, it is imperative to account for LPS contamination in experiments utilizing plasmid DNA for gene transfer, especially in experimental models of immunity and inflammation.